The methods for preparing sulfated compounds reported in the prior art generally induce cleavage of the chains of the polymers during the O or N sulfonation and, thus, cause the formation of potentially toxic reactional residues. It would therefore be advantageous to avoid this drawback by enabling the controlled addition of sulfonate groups under controlled conditions such that the structural integrity of the initial polymer, e.g., an oligosaccharide, is not altered.
Known in the prior art are functionalized polymers with anticoagulant properties and notably a family of derivatives obtained by substitutions on a dextran (D) chain of the carboxymethyl (CM), methyl-benzylamide (B) and methyl-benzylamide sulfonate (S) groups, designated by the abbreviation CMDBS. The methods for the preparation of these polymers were described in particular in the French Patent No. 2,461,724, as well as in U.S. Pat. No. 4,740,594. Neither of these patents provides a precise analysis of the resultant polymeric structures and the methods described do not enable providing evidence that the resultant products are homogeneous. The experimental conditions of the sulfonation reaction described in these patents promote binding of the sulfones to the free OH groups of the glucoside residue and the analysis presented does not enable the clear demonstration of the presence of benzylamine sulfonate.
Other members of the CMDBS family, designated HBGF for “Heparin Binding Growth Factor”, were described as cicatrization agents in U.S. Pat. No. 5,693,625. HBFGPPs were also described for their properties of stimulating the repair and regeneration of lesions induced in muscle tissues (French Patent No. 2,718,024), nervous tissues (French Patent No. 2,718,026) and digestive tract tissues (French Patent No. 2,718,023) as well as for their anti-inflammatory properties (French Patent No. 2,718,025). These patents established a series of functional criteria for screening among all the biocompatible polymers those that respond to the following four functional properties:                protecting heparin binding growth factors (HBGFs) like the fibroblast growth factors such as FGF1 & 2 or transforming factors such as TGF-β against proteolytic degradations as well as potentiating their biological activities in a series of tests on cell cultures;        having an anticoagulant activity of less than 10 international units per mg;        inhibiting the activity of leukocyte elastase under physiological conditions; and        inhibiting the activity of plasmin under physiological conditions.        
French Patent Application No. 98/08309 reports the structure of a family of polymers, designated RGTA for “ReGeneraTing Agent”, having the properties of the HBGFPPs and describes a method for their preparation and their properties. The RGTAs have antifibrotic effects notably enabling improvement in the quality of cutaneous cicatrices, antioxidant effects notably for treating the deleterious effects of free radicals (after ionizing radiation or during the oxidative stress induced by ischemia) and properties of regulating tissue homeostasis notably of bone masses. These properties supplement those described for the HBGFPPs.
The polymers described in French Patent Application No. 98/09309 correspond to the following formula:AaXxYy  (I)in which:                A is a monomer; X represents an RCOOR′ group; Y represents an O- or N-sulfonate group bound on A and responding to either of the following formulas: ROSO3R′ or RNSO3R′; the R groups are aliphatic hydrocarbon chains optionally branched and/or unsaturated and which can contain one or more aromatic rings with the exception of benzylamine and benzylamine sulfonate; R′ represents a hydrogen atom or a cation; a represents the number of monomers; x represents the level or degree of substitution of the A monomers by the X groups; y represents the level or degree of substitution of the A monomers by the Y groups.        
Dextran derivatives such as CMDS are among the polymers described in French Patent Application No. 98/09309. Polymers of the CMDS type have also been described as anticoagulants (Maiga et al., Carbohydrate Polymers, 1997, 32, 89-93).
In these examples of the prior art, the synthesis methods described do not make it possible to obtain products in accordance with the criteria of sufficient reproducibility assuring the maintenance of molecular integrity and the absence of contaminants. In fact, although the methods for the substitution of carboxyl groups are widely described in the prior art and make possible controlled and managed substitution ensuring sufficient reproducibility, the sulfonation methods are more difficult to control.
The sulfonation methods are performed at a very acid pH value which does not enable preservation of the integrity of the polymer chain especially if this chain is constituted of natural sugars. Moreover, these sulfonation conditions lead to decarboxylations that are very difficult to control.
Thus, numerous studies in the prior art describe methods enabling the sulfatation of polysaccharides of type Aa. Thus, for example, dextran sulfate (or O-sulfonate) (DS), carboxymethyl dextran sulfate (CM-D-S) (or O-sulfonate) and other sulfated (or O-sulfonated) oligosaccharides such as xylan and starch were sulfated (or O-sulfonated) by methods of the type described in U.S. Pat. No. 4,814,437. Among these methods, those proposing sulfonation of polysaccharides using strong acids as sulfonation agents were reported by numerous authors. For example, U.S. Pat. Nos. 4,740,594 and 4,755,379, and French Patent No. 2,772,382 describe a treatment with chlorosulfonic acid using dichloromethane (CH2Cl2) as a solvent for the production of molecules of the CM-D-S and DM-D-B-S type. This method was already widely described in these older studies because the same acid was used for the synthesis of DS with pyridine as solvent of a basic nature (Ricketts, Biochem J, 51, 210-233, 1952). Sulfuric acid and sulfonic acid in the presence of formamide were also used for the preparation of DS at low temperatures as described in U.S. Pat. Nos. 3,498,972 and 3,141,014. It is known that these strongly acid reaction conditions induce partial degradation of the products even in the presence of solvents of a basic nature or at low temperatures. They lead to a noteworthy fragmentation of the macromolecular chain in the case of polymer products and a partial hydrolysis of certain functional groups contained in the molecules to be sulfonated. Many improvements have been introduced regarding these strong acidity conditions involving the use of better buffered media or media of lower acidity. Thus, the use of sulfur trioxide (SO3) complexes as sulfonation agents less severe than sulfuric and sulfonic acids has already been introduced (Archives of Biochemistry and Biophysics, 95, 36-41, 1961; Tetrahedron Letters, 29, 7, 803-806, 1988; J. Chem. Soc. Perkin Trans. 1, 157, 1995). Many complexes of the SO3 amine type have been made available commercially such as SO3-ME3N (trimethylamine), SO3-Et3N (triethylamine), SO3-pyridine and SO3-piperidine. These reagents are used in an anhydrous medium in solvents such as DMF, formamide and DMSO. Although these methods are used for the production of sulfated polysaccharides, especially those derived from dextran, functionalized by ester, ether or amid groups, they have major drawbacks because they lead to:                random fragmentation of the macromolecular chain of the polymers, albeit in lower proportions than with sulfonic acids;        partial hydrolysis of the functional groups already present on the polymers cited above; and        generation of secondary products by formation of amines which contaminate the preparations and which have been shown to be toxic after inoculation in vivo (Brain Res, 16, 208-2, 473-478, 1981).        
These techniques have been still further improved to eliminate to the maximum extent possible the toxic residues as well as to propose for pyridine the method described in U.S. Pat. No. 4,814,437. In fact, the SO3-amines used in that method are reactive with the aldehyde groups present on the reducing end of most of the polysaccharides, notably in dextran, and the bond formed in this manner is covalent and thus permanent.
The disadvantage of the use of complexes of the SO3-amine type was also overcome by employing SO3-amine type complexes such as SO3-DMF and SO3-FA. For example, the salts of DS, more particularly the sodium salts, are presently prepared as described in U.S. Pat. No. 4,855,416 by O-sulfonation of dextran by an SO3 complex associated with formamide (SO3-FA) with formamide as solvent. That method comprises formation in situ of the SO3-FA complex and then its reaction with dextran. The strong reactivity of the SO3-FA complex induces a strong acidity of the reactional medium despite the use of an inert atmosphere (N2) and anhydrous solvents. This fact also leads to the degradation of the macromolecular chain.
Another widely used non-animated complex is SO3-DMF. It was in fact proposed in French Patent No. 2,772,382 for the synthesis of sulfated polysaccharides of the CM-D-S and CM-D-B-S type. However, the SO3-DMF complex, like the SO3-FA complex, produces a strong acidity of the reaction medium, which also leads to fragmentation of the macromolecular chain and the hydrolysis of the labile hydrolysis groups resulting in a loss of control of the synthesis and the final products. This method is thus as imperfect as the other methods in the case of the preparation of high-molecular-weight polyanionic polymers which it is not desired to cleave in a random, uncontrolled manner.
It was with this desire to improve the synthesis methods that the protocols for producing CMDBS and CMDS were described in French Patent No. 97/15702. According to French Patent Application No. 97/15702, this improvement enables on the one hand production of dextran derivatives having a greater homogeneity of molecular weight and on the other hand control of the substitution rates thus ensuring a greater homogeneity of the structures and thus a better definition. However, like the prior art, the method described in French Patent Application No. 97/15702 is based on the sulfonation of polysaccharides using an SO3-amine (DMF, pyridine or triethylamine) and the examples presented are exclusively obtained with SO3-pyridine. This method moreover leads to formation of residual traces of pyridine, the human toxicity of which is well known, and does not appear to enable the absence of formation of fragments of the polysaccharide chain.